HIV/AIDS is a major cause of global morbidity and mortality. Direct killing of infected CD4+ T cells and evasion of immune responses all underlie the pathogenesis of AIDS. Viral proteins that mediate immune dysfunction and evasion include the membrane-associated myristoylated Nef protein, which is also critical for viral replication and virulence. Nef mechanisms of immune evasion have not been fully delineated, but include downregulation of major immune cell receptors CD4 and MHC class I. CD4 is the major T cell receptor for HIV and is critical for helping both cell mediated and humoral immunity. CD4 downregulation therefore leads to severe immune dysfunction. MHC class I receptors are critical for presentation of viral antigens on infected cells that are then recognized by CD8 killer cells. Loss of MHC-I (HLA-I) density at the cell surface will allow HIV infected cells to escape immune-surveillance. [unreadable] Genetic and biochemical studies have suggested two different mechanisms for the CD4 and HLA-I downregulation induced by Nef. In the previous reporting period, we showed that both the recycling cell surface receptor and the nascent receptor in transit to the plasma membrane were susceptible to intra-cellular retention and degradation by HIV-1 Nef. In contrast, AP-2 mediated enhanced endocytosis constituted the predominant mechanism for SIV Nef induced downregulation of CD4. [unreadable] Two mutually exclusive mechanisms have been proposed for Nef induced HLA-I downmodulation: 1) a multi-step hierarchical dysregulation of PACS-1 and PI3K dependent ARF6 endocytic pathway or 2) by AP-1 vesicle dependent misrouting of newly synthesized MHC-1 complex from the TGN to the endolysosomal pathway for degradation. During the current reporting period, we evaluated HIV-1 and SIV Nef proteins for their effect on endogenous HLA-I (s) or plasmid expressed A2 allele in epithelial cells, PBLs or T cell lines. By using genetic inhibitors of vesicular traffic or siRNA knockdown of specific adapter subunits of AP1, AP2 or AP3 vesicles, we found Nef induced a delay & misrouting of HLA-I during both antero- & retrograde itineraries. In both primary cells and continuous cell lines, HIV-1 and SIV Nef alleles induced a delay in receptor recycling that was reversed only to a modest extent by genetic inhibitors of vesicular endocytosis. However, all the Nef proteins rerouted de novo synthesized HLA-I (A2) from TGN to lysosomes in an AP1 vesicle dependent manner. Furthermore, we observed that the Nef mutants previously reported to lack the HLA-I downregulation were only partially defective for this property. In sum, our findings indicate that different Nef alleles disrupt AP1 and to a lesser extent AP3 vesicular pathways in subverting both antero- and retrograde trafficking MHC-I alleles.[unreadable] [unreadable] Human chemokine and chemokine receptor dysfunction are associated with numerous acute and chronic diseases such as asthma and arthritis and infectious diseases including AIDS and TB. HIV infection induces defects in lymphocyte homing, DC and macrophage maturation, T lymphocyte differentiation and cytokine and chemokine secretion. And some members of the human chemokine receptor family also serve as coreceptors for HIV entry. Our understanding of this feedback controlled signaling system in health and disease will enable us to understand viral pathogenesis and help us develop novel therapeutic interventions.[unreadable] Chemokine receptors constitute a sub-class in GPCR (the G-protein coupled receptors) superfamily, which is phylogenetically conserved from yeast to primates and regulate diverse biological processes besides leukocyte chemotaxis. In previous years a) we identified the structural requirements of CC and CXC chemokine receptors for the biological function and HIV usage; b) investigated how naturally occurring CCR5 mutants impaired for surface expression impact the physiological function of the wt receptor and its HIV usage in hetrozygotes; c) addressed the mechanistic differences between the CCR5 and CXCR4 receptors in the agonist-driven receptor signaling, and trafficking, and d) showed that in primary leukocytes, agonist driven cell migration and receptor internalization are separable processes. [unreadable] Leukocyte migration towards chemoattractants requires plasma membrane cholesterol and lipid raft integrity. In the current reporting period, we showed that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12), while early chemoattractant signaling events such as G-protein activation and intracellular calcium flux were unaffected. Cholesterol depletion also did not affect endocytosis of agonist-occupied receptors and the signaling pathways such as the activation of MAPK, Hck and Fgr linked to receptor endocytosis. These data suggested that while spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane, events linked to receptor trafficking can occur in the absence of such reorganization.